Note: Descriptions are shown in the official language in which they were submitted.
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RELAXIN-LIKE FACTOR
AND METHODS AND USES THEREOF
A portion of the work set forth herein was
supported by grants NIHGMS-48829 and NSF MCB-9406656
= .
and by the Medical University of South Carolina.
1. INTRODUCTION
The present invention relates to a relaxin-like
factor, its derivatives or analogs, and uses thereof.
The present invention further relates to compositions
and formulations comprising a relaxin-like factor, its
derivatives or analogs, and relaxin wherein such
composition exhibits an additive or synergistic
effect.
2. BACKGROUND OF THE INVENTION
A family of hormones, comprising insulin,
insulin-like growth factors (I and II), bombyxin,
molluscan insulin-related peptide and relaxin, has
been identified and designated as "insulin-related."
Blundell and Humbel, 1980, Nature 287:781-787;
BUllesbach and Schwabe, 1991, J. Biol. Chem.
266:10754-10761. The proteins comprising this family
of hormones represents a group of polypeptides having
homologous primary and secondary structure but
divergent biological functions.
Relaxin has been purified from a variety of
species including porcine, murine, equine, shark,
tiger, rat, dogfish and human. In the human, relaxin
is most abundantly found in the corpora lutea (CL) of
pregnancy. Mature human relaxin is a hormonal peptide
of approximately 6000 daltons which facilitates the
birth process by remodelling the reproductive tract
before parturition. More specifically, relaxin
appears to mediate the restructuring of connective
tissues in target organs to obtain the required
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changes in organ structure during pregnancy and
parturition. See, Hisaw, 1926, Proc. Soc. Exp. Biol.
Med. 23:661-663; Schwabe, et al., 1977, Biochem.
Biophys. Res. Comm. 75:503-570; James, et al., 1977,
Nature, 267:544-546. A concise review of relaxin was
provided by Sherwood, D. in The Physiology of =
Reproduction, Chapter 16, "Relaxin", Knobil, E. and
Neill, J., et al. (eds.), (Raven Press Ltd., New
York), pp. 585-673 (1988).
While predominantly a hormone of pregnancy,
relaxin has also been detected in the non-pregnant
female as well as in the male. Bryant-Greenwood,
1982, Endocrine Reviews 3:62-90; Weiss, 1984, Ann.
Rev. Physiol. 46:43-52.
Two human gene forms encoding for human relaxin
have been identified, (H1) and (H2). Hudson, et al.,
1983, Nature 301 628-631; Hudson, et al., 1984, EMBO
J., 3:2333-2339; and U.S. Patents Nos. 4,758,516 and
4,871,670. Only one of the gene forms (H2) has been
found to be transcribed in CL. It remains unclear
whether the (Hl) form is expressed at another tissue
site, or whether it represents a pseudo-gene. When
synthetic human relaxin (H2) and certain human relaxin
analogs were tested for biological activity, the tests
revealed a relaxin core necessary for biological
activity as well as certain amino acid substitutions
for methionine that did not affect biological
activity. Johnston, et al., in Peptides: Structure
and Function, Proc. Ninth American Pentide Svmposium,
Deber, C.M., et al. (eds.) (Pierce Chem. Co. 1985).
Methods of making relaxin are described in U.S.
Patents No. 4,835,251 and in co-pending U.S. Serial
Nos. 07/908,766 (PCT US90/02085) and 08/080,354 (PCT
US94/0699). Methods of using relaxin in
cardiovascular therapy and in the treatment of =
neurodegenerative diseases are described in U.S.
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Patent No. 5,166,191 and in U.S. Patent No. 5,478,807.
Certain formulations of human relaxin are described in
U.S. Patent No. 5,451,572.
The structure and biological function and
activity of the remaining members of the insulin-
related family have been extensively studied. See,
e.g. Robinson and Fritz, 1981, Biol. Reprod. 24:1032-
1041; Soder, et al., 1992, Endocrinology 131:2344-
2350; Luthman, et al., 1989, Eur. J. Biochem
180(2):259-65; Jhoti, et al., 1987, FEBS Lett.
219:419-425; Smit, et al., 1988, Nature 331:535-538.
Among the structural features shared between relaxin
and the remaining members of the insulin-related
family of hormones are molecular weight, a "two-chain"
structure comprising a B-chain, a connecting C-
peptide, and an A-chain, and the number and
disposition of disulfide links.
Despite these similarities, the proteins
comprising the insulin-related family have been found
to have distinct biological functions and activities.
It has been reported that this distinction is in large
part a consequence of differences between a few type-
specific amino acid residues. For example, the
difference between the glycine in position A14 of
human type II relaxin and the isoleucine in the
equivalent position (A10) of insulin is considered
critical in distinguishing between the biological
activity of the two proteins. Schwabe and Bullesbach,
1994, FASEB J. 8:1-2.
A protein having the structural characteristics
of insulin, insulin-like growth factor (IGF) and
relaxin has been isolated recently from Leydig cells
of the testes. Burkhardt, et al., 1993, Genomics
20:13-19. This protein, designated as a Leydig cell-
specific insulin-like peptide (Ley I-L), has been
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characterized as being "insulin-like" due to the
genomic location of the gene encoding Ley I-L vis a
vis the gene encoding insulin (as compared to the
genomic location of the gene encoding either relaxin
or IGF). Burkhardt, et al., 1993, Genomics 20:13-19.
The Ley I-L protein has been characterized also
as insulin-like, rather than either IGF-like or
relaxin-like, based upon the protein's C-peptide chain
length. More specifically, the C-peptide length of
the Ley I-L protein is 49 amino acids, as compared to
the 35 amino acid length of proinsulin C-peptide, the
twelve amino acid length of the known proIGF C-
peptides and the over one-hundred amino acid C-peptide
length of prorelaxin. Finally, Ley I-L has been
designated insulin-like based on the observation that
the protein is expressed exclusively in prenatal and
postnatal testicular Leydig cells. Burkhardt, et al.,
supra.
On the basis of the protein's similarities to
insulin and the source of such protein, it was
reported that the Ley I-L protein is implicated in
testicular function. Id., Adham, et al., 1993, J.
Bio. Chem. 268(35):26668-6672.
In consultation with the inventors of the present
invention, Tashima, et al., 1995, J. Clin. Endocrinal.
Metab. 80:707-710, have investigated the accuracy of
previous reports providing that the Ley I-L gene was
only expressed in Leydig cells. Specifically,
Tashima, et al. investigated whether the Ley I-L gene
was present and expressed in female reproductive
tissues, the human corpus luteum, trophoblasts, fetal
membranes and breast tissue. As with the case with H2
relaxin, Tashima, et al. determined that the Ley I-L
protein can be found in human corpus luteum and
trophoblast. Unlike H2 relaxin, however, Ley I-L was
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not found to be expressed in fetal membranes, decidua
and breast tissue.
Neither the Burkhardt/Adham group nor the Tashima
group have reported the biological function of the Ley
I-L protein. Thus, while the structure of this
putative Ley I-L protein has been identified, no
correct activity or use was known for this protein
until the present invention, which completed the
discovery of RLF through the identification and proof
of its utility.
3. SUMMARY OF THE INVENTION
The present invention is directed to synthesized
or recombinant compositions derived from the deduced
amino acid and nucleic acid sequences for human Ley I-
L. In one embodiment of the present invention, the
composition comprises the full-length amino acid
sequence for relaxin-like factor (RLF). In another
embodiment of the present invention, the composition
comprises a RLF protein derivative wherein the protein
is shortened at either or both its 3' and 5' ends of
either or both the A- or B-chains. In one embodiment,
the A chain may be as short as fifteen amino acids in
length and the B chain may be as short as thirteen
amino acids in length. In yet further embodiments of
the present invention, the composition is
radiolabelled or represents an analog of RLF having
relaxin-like activity.
The present invention is further directed to the
use of such compounds for the treatment of diseases
and disorders which may be otherwise treated with
relaxin, either alone, or in combination with relaxin
or other relaxin-like agents, and formulations
thereof. In one embodiment of the present invention,
the diseases or disorders are related to the abnormal
expression of collagen and/or fibronectin. More
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specifically, such diseases or disorders include
scleroderma and rheumatoid arthritis. In another
embodiment of the present invention, the diseases
and/or disorders are more generally related to the
activation of one or more biological functions as a
result of binding with the relaxin or RLF receptor. Such diseases and/or
disorders may include
cardiovascular disease, sinus bradycardia,
neurodegenerative or neurologic disease, depression
and hair loss.
The present invention is also related to the use
of RLF, whether labelled or unlabelled, as a tracer
which could then be used to separate, by HPLC, the
different RLF derivatives to yield a carrier-free
tracer, in binding assays, and for RLF receptor
mapping.
4. DESCRIPTION OF THE DRAWINGS
FIG. 1. FIG. 1 depicts the primary structure of
the relaxin-like factor, as compared with the
sequences of human relaxin and insulin wherein the
relative positions of the B-chain arginines in RLF, as
compared to relaxin, is highlighted.
FIG. 2. FIG. 2 depicts a schematic of the site-
directed sequential disulfide link formation.
Specifically, the schematic provides information
regarding: 1) trifluoroacetic acid (TFA) deprotection;
2) oxidation of the thiols using DMSO/50 mM NH4HCO3
(1:2 v/v); 3) HF-deprotection of Cys(4-methylbenzyl);
4) combination of A and B chain pH 4.5 in 8 M
guanidinium chloride; 5) formation of the third
disulfide link by reaction with iodine in 70% acetic
acid; 6) liberation of the tryptophan side chain with
10% piperidine; 7) reduction of methionine sulfoxide
with a 33 fold excess of NH4I in 90% TFA.
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FIG. 3. FIG. 3 depicts the HPLC record of the
purified RLF. Chromatography was performed on
Synchropak RP-P (4.1 x 250 mm) using a linear gradient
from 20 - 50% in 30 min (A:0.1% TFA in H20 and B:0.1%
TFA in 80% acetonitrile) at a flow rate of 1 ml/min.
FIG. 4. FIG. 4 depicts a comparison of the CD
spectra of human relaxin, human RLF, and porcine
relaxin.
FIG. 5. FIG. 5 depicts the elution record of the
HPLC separation of an RLF tracer preparation. The
largest peak is unmodified RLF and the shaded region
is the major radioactive peak used as tracer.
Chromatography was performed on Aquapore 300 (2.1 mm x
30 mm) using a linear gradient from 23% B to 34% B
over 60 min (A: 0.1% TFA in H20 and B: 0.1% TFA in 80%
acetonitrile) at a flow rate of 0.1 ml/min.
FIG. 6. FIG. 6 depicts the tissue distribution
of RLF receptors in female estrogen primed mice as
measured in vitro in a receptor-binding assay.
FIG. 7. FIG. 7 depicts the bioactivity of an
increasing amount of relaxin in the presence and
absence of 5 g of RLF per animal. The increase in
symphyseal width was easily recognized.
FIG. 8. FIG. 8 depicts the bioactivity of an
increasing amount of RLF in the presence of a uniform
amount of human relaxin again shows relaxin
enhancement.
FIG. 9. FIG. 9 depicts a comparative bioassay of
relaxin, RLF, and an optimal dose of both. RLF alone
does not cause symphyseal widening but the high dose
of the mixture still improves upon the high dose of
relaxin alone.
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5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Definitions
As used in the present specification, the following words and phrases are
generally intended to
have the meanings as set forth below, except to the
extent that the context in which they are used
indicates otherwise.
"Optional" or "optionally" means that the
subsequently described event or circumstance may or
may not occur, and that the description includes
instances where said event or circumstance occurs and
instances in which it does not.
The term "effective amount" means a dosage
sufficient to provide treatment for the disease state
being treated. This will vary depending on the
patient, the disease and the treatment being effected.
The term "relaxin" means human relaxin, including
full length relaxin or a portion of the relaxin
molecule that retains biological activity [as
described in U.S. Patent No. 5,023,321, preferably
recombinant human relaxin (H2)] and other active
agents with relaxin-like activity, such as agents that
competitively displace bound relaxin from a receptor.
Relaxin can be made by any method known to those
skilled in the art, preferably as described in U.S.
Patents No. 4,835,251 and in co-pending U.S. Serial
Nos. 07/908,766 (PCT US90/02085) and 08/080,354 (PCT
US94/0699).
5.2. Relaxin-Like Factor: Structure And Activity
RLF shares primary and secondary structural
homology with relaxin, insulin and the other members
of the insulin-related family of hormones. As
reported previously, RLF is structurally closer to
insulin than relaxin. The deduced primary structure
of RLF is set forth at FIG. 1.
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Contrary to early reports, however, the
biological function and activity of RLF is similar to
relaxin and distinct from insulin. For example,
notwithstanding the shift in amino acid sequence of
the receptor interacting region between RLF and
relaxin, RLF interacts with the mouse brain receptor
to which relaxin binds.
A basis for the present invention is the
inventors' unexpected discovery that the previously
isolated but uncharacterized RLF protein binds
specifically to crude membrane preparations of mouse
uterus and brain and shows crossreactivity with the
relaxin receptor, but not the insulin receptor.
The deduced amino acid sequence for RLF would
have predicted an opposite result because the critical
Arg XXX Arg sequence in RLF is offset toward the C-
terminal end of the B chain by exactly one turn of the
helix. Thus, although RLF projects the arginines at
nearly right angles away from the molecular surface in
the manner of relaxin, one would expect that shifting
the whole receptor-binding site would present quite a
different binding environment to the receptor.
Notably, although RLF binds to the relaxin
receptor, it does not appear to competitively bind to
the relaxin receptor, vis a vis relaxin, except at
higher concentrations. Rather, RLF appears to
stimulate relaxin response. Thus, RLF can play an
important supportive role for the relaxin action in
humans. In addition, preliminary experiments suggest
that RLF plays a role independent of relaxin in the
male gonads.
In addition, although relaxin-like activity has
historically been considered in terms of softening the
pubic and cervical ligaments in preparation for
parturition, it has also been shown to directly effect
cells outside of the reproductive system. For
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example, consistent with relaxin, RLF may also be
instrumental in inhibiting collagen and/or fibronectin
overexpression and diseases related thereto (e.g.
scleroderma)
Moreover, although RLF possesses relaxin-
enhancing properties, as described herein, RLF possess
independent and potentially additional biological
activity.
5.3. RLF Derivatives And Analogs
Following the present identification of RLF
as a protein having relaxin-like (rather than insulin-
like) activity, to the extent that RLF shares primary
and secondary homology to relaxin, as well as insulin,
identification of biologically active derivatives and
analogs of relaxin evidences the identity of
biologically active RLF derivatives and analogs.
Active relaxin analogs and derivatives have been
identified to include, for example, shortening either
or both the 5' and 3' end of the protein. See e.g.,
U.S. Patent No. 5,023,321. The present invention is
therefore directed to biologically active RLF
derivatives wherein the 5' and/or 3' end of the
protein has been shortened. See, above referenced
patents.
Importantly, it has been observed in human
relaxin that the arginines at positions B13 and B17
and potentially the amino acids of the first helix
turn in the midregion of the B-chain (Arg-Glu-Leu-Val-
Arg) are necessary or important to relaxin activity.
Other RLF analogs and derivatives may be obtained
using known techniques and this structural information
regarding relaxin.
Whether the RLF derivative or analog possesses relaxin-like activity and/or
utility may be determined
using assays known in the art for detecting relaxin
activity. For example, bioassays used for the
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measurement of active relaxin during pregnancy and
non-pregnancy, as described in Steinetz et al., 1960,
Endocrinology 67:102-115 and Sarosi et al., 1983,
American Journal of Obstetrics and Gynecology 145:402-
405, may be used.
Similarly, specific immunoassays to detect for
the presence of proteins having relaxin-like activity
may also be used. See e.g., Sherwood et al., 1975,
Endocrinology 107:691-696; O'Bryne and Steinetz, 1976,
Proceedings of the Society for Experimental Biology
and Medicine 152:272-276. The presence and activity
of synthetic analogs of human relaxin comprising one
or more accessible tyrosines (permitting direct
iodination) may also be tested using a
radioimmunoassay (RIA). Eddie et al., 1986, Lancet
1=1344-1346.
Each of the above-described assays, however, are
limited in their application. Thus, as set forth
below and as described in more detail in a co-pending
application, filed concurrently herewith and entitled
"Relaxin Diagnostic Assays And Kits," additional
assays may also be used to assay RLF to determine the
protein's activity and preferred applications.
5.4. Production Of RLP
RLF may be produced using techniques
previously disclosed as useful in producing relaxin
and insulin. For example, the cDNA for RLF disclosed
in Burkhardt, et al., 1994, Genomics 20:13-19 and
'Adham, et al., 1994, J. Biol. Chem. 268:26668-26672
may be used to recombinantly produce RLF according to
processes previously described as useful in
recombinantly manufacturing relaxin (e.g., U.S.
Patents Nos. 4,758,516, 4,871,670, 4,835,251 and co-
pending U.S. Serial Nos. 07/908,766 (PCT US90/02085)
and 08/080,354 (PCT US94/0699)). Similarly, such
sequence information may be used to synthesize RLF
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according to the methods of Bullesbach and Schwabe,
1991, J. Biol. Chem. 266:10754-10761, for synthesizing
relaxin.
Derivatives and analogs of RLF also may be
synthesized according to the methods of Bullesbach and
Schwabe, supra. Alternatively, such derivatives and
analogs may be produced recombinantly using, for
example, site directed mutagenesis techniques as set
forth in Tsurushita, et al., 1988, Gene 62:135-139.
Relaxin, for use in compositions containing RLF,
may be obtained using any number of readily available
techniques.
For example, naturally-occurring relaxin may be
purified from a variety of species including porcine,
murine, equine, shark, tiger, rat, dogfish and human.
In the human, relaxin is found in most abundance in
the corpora lutea (CL) of pregnancy.
Relaxin may also be synthesized according to the
techniques described above, with respect to RLF, or
alternatively, recombinantly, by relying upon the
disclosed nucleic acid sequences and deduced amino
acid sequences for relaxin. In humans, two gene forms
encoding for human relaxin have been identified, (H1)
and (H2) and their use to recombinantly manufacture
relaxin, and preferably relaxin (H2), have been
described. Hudson, et al., 1983, Nature 301 628-631;
Hudson, et al., 1984, EMBO J., 3:2333-2339; and U.S.
Patents Nos. 4,758,516 and 4,871,670. Methods of
making relaxin are also described in U.S. Patents No.
4,835,251 and in co-pending U.S. Serial Nos.
07/908,766 (PCT US90/02085) and 08/080,354 (PCT
US94/0699).
Notably, when synthetic human relaxin (H2) and certain human relaxin analogs
were tested for
biological activity, the tests revealed a relaxin core
necessary for biological activity as well as certain
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amino acid substitutions for methionine that did not
affect biological activity. Johnston, et al., in
Peptides: Structure and Function, Proc. Ninth
American Peptide Symposium, Deber, C.M., et al. (eds.)
(Pierce Chem. Co. 1985).
5.5. Indications/Methods Of Use
In vitro, proteins having relaxin-like
activity decrease collagen synthesis by human dermal
and synovial fibroblasts upregulated to overexpress
collagen with transforming growth factor-beta (TGF-
beta) or interleukin-1, and by fibroblasts
constitutively overexpressing collagen obtained from
scleroderma patients. For example, relaxin decreases
collagen accumulation in vivo in two rodent models of
fibrosis. Relaxin or relaxin-like proteins also
increase the secretion of the collagenolytic
metalloproteinase, collagenase, and also down-
regulates the expression of the metalloproteinase
inhibitor, tissue inhibitor of metalloproteinases.
Relaxin has been implicated consequently in the
treatment and diagnosis of various diseases and
disorders. For example, studies provide evidence that
relaxin is effective in the treatment of scleroderma,
sinus bradycardia, cardiovascular disease
neurodegenerative and neurologic disorders, hair loss,
depression. See e.g., U.S. Patent No. 5,166,191; U.S.
Patent No. 5,478,807; U.S. Applications entitled
"Method For Treatment Of Hair Loss" and "Method For
Treatment Of Depression", both of which are filed
concurrently herewith. Evidence also suggests the use
of relaxin in diseases and disorders related to the
abnormal expression of collagen or fibronectin, such
as scleroderma or rheumatoid arthritis.
As provided herein, RLF possesses relaxin-like
biological activity and is therefore similarly
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implicated in the above described diseases. Moreover,
to the extent that RLF is also shown to enhance the
activity of relaxin, RLF, as administered in
combination with relaxin or another agent, is also
indicated for the treatment of the above-identified
diseases.
Additionally, as more fully discussed in the U.S.
Application entitled "Relaxin Diagnostic Assays and
Kits," filed concurrently herewith, diagnostic assays
for determining the predisposition or presence of
prostate, breast, testicular, ovarian and other
cancers having common stem cell heritage, which rely
on detecting the presence of relaxin may also be
adjusted to rely upon the detection of RLF. Such
assays can also be used to follow-up on tumor
metastases after ablation of cancer.
5.6. Pharmaceutical Dosage Requirements,
Formulations And Routes Of Administration
The following dosage requirements,
formulations and routes of administration for RLF are
discussed below:
5.6.1. Effective Dosage.
Pharmaceutical compositions
suitable for use in the present invention include
compositions wherein the active ingredients are
contained in an effective amount to achieve its
intended purpose. More specifically, a
therapeutically effective amount means an amount
effective to prevent development of or to alleviate
the existing symptoms of the subject being treated.
Determination of the effective amounts is well within
the capability of those skilled in the art, especially
in light of the detailed disclosure provided herein.
For any compound used in the method of the
invention, the therapeutically effective dose can be
estimated initially from cell culture assays. For
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example, a dose can be formulated in animal models to
achieve a circulating concentration range that
includes the IC50 as determined in cell culture. Such
information can be used to more accurately determine
useful doses in humans.
A therapeutically effective dose refers to that
amount of the compound that results in amelioration of
symptoms or a prolongation of survival in a patient.
Toxicity and therapeutic efficacy of such compounds
can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals,
e.g., for determining the LD50 (the dose lethal to 50%
of the population) and the ED50 (the dose
therapeutically effective in 50% of the population).
The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as
the ratio between LD50 and ED50. Compounds which
exhibit high therapeutic indices are preferred. The
data obtained from these cell culture assays and
animal studies can be used in formulating a range of
dosage for use in human. The dosage of such compounds
lies preferably within a range of circulating
concentrations that include the ED50 with little or no
toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route
of administration utilized. The exact formulation,
route of administration and dosage can be chosen by
the individual physician in view of the patient's
condition. (See e.g. Fingl et al., 1975, in "The
Pharmacological Basis of Therapeutics", Ch. 1 p1).
Dosage amount and interval may be adjusted
individually to provide plasma levels of the active
moiety which are sufficient to maintain the relaxin-
like activity and effects.
Administration of RLF, with relain or other
active agents, can be via any of the accepted modes of
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administration for agents that serve similar
utilities, preferably by systemic administration.
While human dosage levels for treating many of
the above-identified relaxin-related diseases or
disorders have yet to be optimized for RLF,
administered alone or in combination with relaxin,
generally, a daily dose is from about 0.1 to 500.0
Ag/kg of body weight per day, preferably about 6.0 to
200.0 g/kg, and most preferably about 12.0 to 100.0
g/kg, depending on whether RLF is administered alone
or in combination with relaxin. Generally it is
sought to obtain a serum concentration of RLF, alone
or in combination with relaxin, approximating or
greater than normal circulating levels in pregnancy,
i.e., 1.0 ng/ml, such as 1.0 to 20 ng/ml, preferably
1.0 to 20 ng/ml.
For administration to a 70 kg person, the dosage
range would be about 7.0 g to 3.5 mg per day,
preferably about 42.0 g to 2.1 mg per day, and most
preferably about 84.0 to 700.0 g per day. The amount
of RLF administered will, of course, be dependent on
the subject and the severity of the affliction, the
manner and schedule of administration and the judgment
of the prescribing physician. One treatment regimen
can employ a higher initial dosage level (e.g., 100 to
200 g/kg/day) followed by decreasing dosages to
achieve steady relaxin or relaxin-like serum
concentration of about 1.0 ng/ml. Another treatment
regimen, particularly postpartum depression, entails
administration of an amount of relaxin sufficient to
attain normal pregnancy levels of relaxin (about 1.0
ng/ml) followed by gradual decreasing dosages until
relaxin serum levels are no longer detectable (e.g.
less than about 20 picograms/ml), optionally
discontinuing treatment upon reaching that dosage
level.
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In employing RLF, either alone or in combination
with relaxin, for treatment of the above conditions,
any pharmaceutically acceptable mode of administration
can be used. RLF can be administered either alone or
in combination with other pharmaceutically acceptable
excipients, including solid, semi-solid, liquid or
aerosol dosage forms, such as, for example, tablets,
capsules, powders, liquids, gels, suspensions,
suppositories, aerosols or the like. Relaxin can also
be administered in sustained or controlled release
dosage forms (e.g., employing a slow release
bioerodable delivery system), including depot
injections, osmotic pumps (such as the AlzetTM implant
made by Alza), pills, transdermal (including
electrotransport) patches, and the like, for prolonged
administration at a predetermined rate, preferably in
unit dosage forms suitable for single administration
of precise dosages. The compositions will typically
include a conventional pharmaceutical carrier or
excipient and RLF. In addition, these compositions
may include other active agents, carriers, adjuvants,
etc.
In a preferred aspect of the invention, a
sustained/controlled release RLF formulation was a
selectively permeable outer barrier with a drug
dispensing opening, and an inner RLF-containing
portion designed to deliver dosage of RLF
progressively diminished as a predetermined rate (e.g.
containing about 30 mg of RLF in a matrix for delivery
of initially about 500 ug per day diminishing at a
rate of 10 pg per day.
In another preferred aspect of the invention, a
sustained/controlled release RLF formulation has a
selectively permeable outer barrier with a drug
dispensing opening, a first inner relaxin-containing
portion designed for steady state release of relaxin
17
CA 02224080 2008-10-21
at a therapeutically effective daily dosage (e.g.
containing about 50 mg of relaxin in a matrix for
continuous delivery of about 500 pg per day), and a
second inner RLF-containing portion designed to
deliver a dosage of RLF progressively diminishing at a
predetermined rate (e.g. containing about 3 mg of
relaxin in a matrix for delivery of initially about
500 pg per day diminishing at a rate of 50 pg per day)
commencing upon exhaustion of the relaxin from the
first inner portion.
Generally, depending on the intended mode of
administration, the pharmaceutically acceptable
composition will contain about 0.1% to 90%, preferably
about 0.5% to 50%, by weight of RLF, either alone or
in combination with relaxin, the remainder being
suitable pharmaceutical excipients, carriers, etc.
Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in this
art; for example, see Remington's Pharmaceutical
Sciences, Mack Publishing Company, Easton,
Pennsylvania, 15th Edition, 1975. The formulations of
human relaxin described in U.S. Patent No. 5,451,572
are particularly preferred.
In cases of local administration or selective
uptake, the effective local concentration of the drug
may not be related to plasma concentration.
The amount of composition administered will, of
course, be dependent on the subject being treated, on
the subject's weight, the severity of the affliction,
the manner of administration and the judgment of the
prescribing physician.
5.6.2. Routes Of Administration.
Suitable routes of administration
may, for example, include oral, rectal, transmucosal,
or intestinal administration. Parenteral
administration is generally characterized by
18
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WO 96/40186 PCT/US96/07399
injection, either subcutaneously, intradermally,
intramuscularly or intravenously, preferably
subcutaneously. Injectables can be prepared in
conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as
emulsions. Suitable excipients are, for example,
water, saline, dextrose, glycerol, ethanol or the
like. In addition, if desired, the pharmaceutical
compositions to be administered may also contain minor
amounts of non-toxic auxiliary substances such as
wetting or emulsifying agents, pH buffering agents,
solubility enhancers, and the like, such as for
example, sodium acetate, sorbitan monolaurate,
triethanolamine oleate, cyclodextrins, and the like.
The percentage of RLF and/or relaxin contained in
such parenteral compositions is highly dependent on
the specific nature thereof, as well as the needs of
the subject. However, percentages of active
ingredient of 0.01% to 10% in solution are employable,
and will be higher if the composition is a solid which
will be subsequently diluted to the above percentages.
Preferably the composition will comprise 0.2-2% of the
RLF, alone or in combination with relaxin in solution.
A more recently devised approach for parenteral
administration employs the implantation of a
slow-release or sustained-release system, such that a
constant level of dosage is maintained. See, e.g.,
U.S. Patent No. 3,710,795.
Alternately, one may administer the compound in a
local rather than systemic manner, for example, via
injection of the compound directly into a solid tumor,
often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a
targeted drug delivery system, for example, in a
liposome coated with tissue-specific antibody. The
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liposomes will be targeted to and taken up selectively
by the tissue.
5.6.3. Composition/Formulation.
The pharmaceutical compositions of
the present invention may be manufactured in a manner
that is itself known, e.g., by means of conventional
mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or
lyophilizing processes.
Pharmaceutical compositions for use in accordance
with the present invention thus may be formulated in
conventional manner using one or more physiologically
acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active
compounds into preparations which can be used
pharmaceutically. Proper formulation is dependent
upon the route of administration chosen.
The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection
or continuous infusion. Formulations for injection
may be presented in unit dosage form, e.g., in
ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral
administration include aqueous solutions of the active
compounds in water-soluble form. Additionally,
suspensions of the active compounds may be prepared as
appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils
such as sesame oil, or synthetic fatty acid esters,
such as ethyl oleate or triglycerides, or liposomes.
Aqueous injection suspensions may contain substances
which increase the viscosity of the suspension, such
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as sodium carboxymethyl cellulose, sorbitol, or
dextran. Optionally, the suspension may also contain
suitable stabilizers or agents which increase the
solubility of the compounds to allow for the
preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in
powder form for constitution with a suitable vehicle,
e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal
compositions such as suppositories or retention
enemas, e.g., containing conventional suppository
bases such as cocoa butter or other glycerides.
In addition to the formulations described
previously, the compounds may also be formulated as a
depot preparation. Such long acting formulations may
be administered by implantation (for example
subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be
formulated with suitable polymeric or hydrophobic
materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic
compounds of the invention is a cosolvent system
comprising benzyl alcohol, a nonpolar surfactant, a
water-miscible organic polymer, and an aqueous phase.
The cosolvent system may be the VPD co-solvent system.
VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of
the nonpolar surfactant polysorbate 80, and 65% w/v
polyethylene glycol 300, made up to volume in absolute
ethanol. The VPD co-solvent system (VPD:5W) consists
of VPD diluted 1:1 with a 5% dextrose in water
solution. This co-solvent system dissolves
hydrophobic compounds well, and itself produces low
toxicity upon systemic administration. Naturally, the
proportions of a co-solvent system may be varied
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considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity
of the co-solvent components may be varied: for
example, other low-toxicity nonpolar surfactants may
be used instead of polysorbate 80; the fraction size
of polyethylene glycol may be varied; other
biocompatible polymers may replace polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars
or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for
hydrophobic pharmaceutical compounds may be employed.
Liposomes and emulsions are well known examples of
delivery vehicles or carriers for hydrophobic drugs.
Certain organic solvents such as dimethylsulfoxide
also may be employed, although usually at the cost of
greater toxicity. Additionally, the compounds may be
delivered using a sustained-release system, such as
semipermeable matrices of solid hydrophobic polymers
containing the therapeutic agent. Various of
sustained-release materials have been established and
are well known by those skilled in the art.
Sustained-release capsules may, depending on their
chemical nature, release the compounds for a few weeks
up to over 100 days. Depending on the chemical nature
and the biological stability of the therapeutic
reagent, additional strategies for protein
stabilization may be employed.
The pharmaceutical compositions also may comprise
suitable solid or gel phase carriers or excipients.
Examples of such carriers or excipients include but
are not limited to calcium carbonate, calcium
phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as
polyethylene glycols.
Formulations of RLF may also be administered to
the respiratory tract as a nasal or pulmonary
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inhalation aerosol or solution for a nebulizer, or as
a microfine powder for insufflation, alone or in
combination with an inert carrier such as lactose, or
with other pharmaceutically acceptable excipients. In
such a case, the particles of the formulation may
advantageously have diameters of less than 50 microns,
preferably less than 10 microns. See, e.g., U.S.
Patent No. 5,364,838, which discloses a method of
administration for insulin that can be adapted for the
administration of RLF, alone or in combination with
relaxin in the present invention.
RLF for treatment of such disorders such as
alopecia, may also be administered topically in a
formulation adapted for application to the scalp, such
as a shampoo (e.g., as disclosed in U.S. Patent No.
4,938,953, adapted according to methods known by those
skilled in the art, as necessary for the inclusion of
protein ingredients) or a gel (e.g., as disclosed in
U.S. Patent No. 5,451,572) optionally with increased
relaxin concentrations to facilitate absorption.
For oral administration, the compounds can be
formulated readily by combining the active compounds
with pharmaceutically acceptable carriers well known
in the art. Such carriers enable the compounds of the
invention to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions
and the like, for oral ingestion by a patient to be
treated. Pharmaceutical preparations for oral use can
be obtained solid excipient, optionally grinding a
resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize
23
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starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be .
added, such as the cross-linked polyvinyl pyrrolidone,
agar, or alginic acid or a salt thereof such as sodium
alginate.
Dragee cores are provided with suitable coatings.
For this purpose, concentrated sugar solutions may be
used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or
dragee coatings for identification or to characterize
different combinations of active compound doses.
Pharmaceutical preparations which can be used
orally include push-fit capsules made of gelatin, as
well as soft, sealed capsules made of gelatin and a
plasticizer, such as glycerol or sorbitol. The
push-fit capsules can contain the active ingredients
in admixture with filler such as lactose, binders such
as starches, and/or lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In
soft capsules, the active compounds may be dissolved
or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In
addition, stabilizers may be added. All formulations
for oral administration should be in dosages suitable
for such administration.
For administration by inhalation, the compounds
for use according to the present invention are
conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebulizer,
with the use of a suitable propellant, e.a.,
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dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other
suitable gas. In the case of a pressurized aerosol
the dosage unit may be determined by providing a valve
to deliver a metered amount. Capsules and cartridges
of e.g. gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or
starch.
5.6.4. Packaging
The compositions may, desired,
be presented in a pack or dispenser device which may
contain one or more unit dosage forms containing the
active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The
pack or dispenser device may be accompanied by
instructions for administration. Compositions
comprising a compound of the invention formulated in a
compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and
labelled for treatment of an indicated condition.
Suitable conditions indicated on the label may include
treatment of depression, sinus bradycardia, hair loss,
neurologic or neurodegenerative diseases, scleroderma,
cardiovascular disease or disorders or diseases
related to uncontrolled or abnormal collagen or
fibronectin formation.
More specific dosage, formulation and methods of
administration may be derived from information
contained in U.S. Patent Nos. 5,166,191, U.S. Patent
No. 5,478,807 and U.S. Patent No. 5,451,572, and co-
pending applications, filed concurrently herewith,
entitled "Method Of Treatment For Hair Loss" and
"Method Of Treatment For Depression".
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6. EXAMPLES
The following preparations and examples are given
to enable those skilled in the art to more clearly
understand and to practice the present invention.
They should not be considered as limiting the scope of
the invention, but merely as being illustrative and
representative thereof.
6.1. RLP Synthesis And Confirmation Of
Synthesized Protein
As described above, RLF may be produced by
isolating the protein from natural sources,
synthesizing the protein based on RLF's deduced amino
acid sequence and recombinantly manufacturing the
protein based upon available cDNA data.
One procedure for synthesizing RLF is as follows:
Materials. L-Amino acid derivatives for peptide
synthesis were purchased either from Bachem Bioscience
(Philadelphia, PA) or Bachem California (Torrance,
CA). Solvents for peptide synthesis and
chromatography were distilled in glass (Burdick and
Jackson; Muscagon, MI), and the chemicals for peptide
synthesis were obtained from Perkin Elmer Applied
Biosystems (Foster City, CA). Other chemicals of
analytical grade were used without further
purification.
Methods. The following method was followed to
synthesize RLF:
Peptide Synthesis: The B chain of the RLF
protein was synthesized by tert=butyloxycarbonyl'-
chemistry using conventional HF-labile side chain-
protecting groups for all three functional amino acids
except cysteines. Cysteine B10 was protected by the
acetamidomethyl group and B23 by the thiol-
protecting/activating group [S-(3-nitro-2-
pyridinesulfenyl)] (CysB23). Methionine was protected
by suifoxide formation, and tryptophan by the
26
SUBSTtTUTE SHEET (RULE 26)
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N(in)formyl group. The synthesis was performed on an
Applied Biosystems peptide synthesizer model 430A on
[4-(oxymethylphenylacetamidomethyl)] resin loaded with
0.4 mmol tertbutyloxycarbonyl-alanine. Deprotection
and removal from the solid support was accomplished by
HF-treatment in the presence of 5% m-cresol. The
crude peptide was extracted with 20% acetic acid and
lyophilized (yield 1.387 g). The B chain was purified
on SephadexTM G50-sf (2.5 cm x 50 cm) in 1 M acetic
acid (yield:840 mg), followed by preparative HPLC on
SynchropakTM RP-P (2.1 cm x 25 cm) in portions of 50 to
70 mg. The mobile phase consisted of 0.1%
trifluoroacetic acid (TFA) in water (solvent A) and
0.1% TFA in 80% acetonitrile (solvent B). The column
was equilibrated in 20% B and the peptide eluted with
a linear gradient of 20% B to 50% B over 1 h at a flow
rate of 5 ml/min (overall yield:233 mg). Amino acid
composition: Thr 2.00 (2); Ser 0.86 (1); Glu 2.90 (3);
Gly 3.28 (3); Ala 2.16 (2); Cys 0.89 (2); Val 3.19
(3); Met 1.22 (1); Leu 1.94 (2); Phe 0.99 (1); His
2.44 (2); Lys 0.96 (1); Arg 3.81 (4).
The A chain (0.25 mmol) was synthesized via Fast-
moc chemistry on an ABI peptide synthesizer (model
430A) on p-benzyloxybenzyl resin. All side chains
were protected by TFA-labile protecting groups except
Cys(All), which was acetamidomethyl-protected, and Cys
(A24) which was protected by the HF-labile p-
methylbenzyl group. The A chain was deprotected with
TFA/thiophenol (10:1 v/v), using 50 mg peptidyl
resin/ml for 90 min at room temperature (5). The TFA
was evaporated and the peptide precipitated with
ether. The precipitate was collected by
centrifugation, the supernatant discarded, and the
pellet washed twice with ether and air-dried. The
peptide was suspended in water, dissolved by the
addition of ammonia, and desalted on SephadexTM G25-m
27
CA 02224080 2008-10-21
in 50 mM NH4HCO3. To the eluate (100 ml) 50 ml of Me2SO
was added in order to accelerate the oxidation of the
intrachain disulfide bond A10-A15 (6). The progress
of oxidation was observed by the Eliman reaction (7).
After completion of the disulfide bond formation the A
chain was dialyzed against water and lyophilized
(yield 372.3 mg). Aliquots of 20 mg were further
purified by preparative HPLC on SynchropakT" RP-P (10
mm x 250 mm), using 0.1% TFA in water for solvent A
and 0.1% TFA in 80% acetonitrile for solvent B. The
column was equilibrated in 30% B and the peptide
eluted with a linear gradient of 30% B to 50% B over
30 min at a flow rate of 3 ml/min (overall yield:
166.5 mg). Amino acid composition: Asp 2.20 (2); Thr
3.00 (3); Ser 0.99 (1); Glu 1.92 (2); Pro 2.25 (2);
Gly 1.06 (1); Ala 4.18 (4); Cys 1.62 (4); Leu 3.60
(4); Tyr 1.82 (2); Arg 0.98 (1).
For chain combination, 33.4 mg (11.3 pmol) of the
A chain (acetamidomethylAlO, 4-methylbenzylA24) was
treated with 4 ml of HF in the presence of 200 }al of
m-cresol for 45 min at 0 C. Thereafter the HF was
evaporated in a stream of nitrogen and the peptide
precipitated with ether. The pellet was collected and
dried over KOH in vacuo for 30 min. The monothiol A
chain was dissolved in 4 ml of 8 M guanidiniumchloride
in 0.1 M acetic acid at pH 4.5 and added to 36.3 mg
(9.6 prnol) of the B chain. The disulfide bond A24/B23
was formed at 37 C for 24 h and the resulting product
separated first on SephadexTM G50-sf in 1 M acetic acid
(column 2.5 cm x 50 cm) (yield 48.7 mg, 78.3%),
followed by preparative HPLC on SynchropakT"' RP-P (10
mm x 250 mm) using 0.1% TFA in water for solvent A and
0.1% TFA in 80% acetonitrile for solvent B. The
column was equilibrated in 30% B and the peptide
eluted with a linear gradient of 30% to 45% B over 30
28
CA 02224080 2008-10-21
min at a flow rate of 3 ml/min (yield: 34.1 mg,
54.80) .
The resulting peptide contained acetamidomethyl
groups in positions Cys All and Cys B10, the N(in)
formyl group in Trp B27, and a sulfoxide in the side
chain of Met B5. For the formation of the third
disulfide bond the peptide (9.3 mg) was dissolved n
water (3.5 ml) and added to a stirred solution
consisting of acetic acid (3.5 ml) 6 N HC1 (19.1 f.[l)
and 3 ml of 50 mM iodine in acetic acid (8). The
reaction was performed at room temperature for 10 min,
quenched with ascorbic acid, and the product was
desalted on SephadexTM G25-sf in 1 M acetic acid and
lyophilized. After purification by preparative HPLC
(conditions as before) (yield: 3.42 mg, 36.8%) the
protein still contained protecting groups in Trp (B27)
and Met (B5).
Complete deprotection was achieved first by
treatment of 11.3 mg of the peptide with 2 ml of
water/piperidine 9:1 (v/v) for 2 min at room
temperature. The base was neutralized with 0.4 ml
acetic acid and the peptide purified by preparative
HPLC, dried (yield:11.0 mg, 97.5%), and 10 mg of
peptide-containing methionine sulfoxide was reduced
with 1 ml of TFA/0.5 M NH4I in water 9:1 v/v for 15 min
at 0 C. Free iodine was reduced with 0.5 M ascorbic
acid in water and the reaction quenched by dilution
with water. The final peptide was recovered by
preparative HPLC (conditions as before) (yield 7.57
mg=75.7%). Amino acid compositions: Asp 2.02 (2), Thr
4.79 (5), Ser 1.77 (2), Glu 4.86 (5), Pro 5.17 (5),
Gly 4.15 (4), Ala 6.09 (6), Cys 3.51 (6), Val 2.86
(3), Met 0.70 (1), Ile 0 (0), Leu 5.74 (6), Tyr 2.12
(2), Phe 0.98 (1), His 2.00 (2), Lys 1.26 (1), Trp
1.00 (1), Arg 5.02 (5). (overall yield 14.9%).
29
CA 02224080 2008-10-21
The mobile phase of all HPLC systems used
consisted of 0.1% trifluoroacetic acid in water
(solvent A) and 0.1% trifluoroacetic acid in 80%
acetonitrile (solvent B).
For preparative HPLC, a WatersTM HPLC system
consisting of two pumps (model 6000A) and gradient
programmer (model 680) was used in combination with a
SynchropakTM RP-P column (C18) (SynChrom, In) and an
UvicordTM S UV (226 nm) monitor (LKB, Bromma Sweden).
Usually 1 to 20 mg of peptide was separated using
linear gradients as indicated.
Analytical HPLC I was performed on AquaporeTM 300
(C8;2.1 mm x 30 mm) using an Applied Biosystems HPLC
model 130A. Separation was achieved with a linear
gradient from 23% to 34% B in 60 min at a flow rate of
0.1 ml/min. The peptide was detected by UV absorbance
at 230 nm.
Analytical HPLC II was performed on SynchropakT"`
RP-P (C18, 4.1 mm x 250 mm) using a WatersTM HPLC
system. Separation was achieved with a linear
gradient from 20% B to 50% in 30 min at a slow rate of
1 ml/min. The peptide was detected by UV absorbance
at 220 nm. The above-described HPLC may also be used
to verify the purity of the RLF, as set forth in FIG.
3.
The primary structure of RLF as synthesized
according to the above procedure, or any other solid
phase methodologies in combination with site-directed
sequential disulfide bond formation (a schematic
depicting said formation is set forth at FIG. 2), is
set forth at FIG. 1.
Protein Confirmation And Verification. The
identity of the synthesized RLF may be confirmed and
verified according to known techniques.
Amino Acid Analyses: Following protein
synthesis and purification, amino acid analyses was
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conducted to confirm the protein's identity. First,
peptides were hydrolyzed in vapor phase 6N HCI
containing 0.1% phenol for 1 h at 150 C. The amino
acids were detected after pre-column modification with
phenylisothiocynate and separation by HPLC (Pico=Tag
system, Waters Millipore).
Bequence Analyses: The identity of the
protein was verified by sequence analysis.
Specifically, such analyses were performed on an ABI
477 A pulsed liquid protein sequencer and an in-line
ABI 120A phenylthiohydantoin analyzer (ABI, Applied
Biosystems, Foster City, CA). Chains were prepared by
reduction of about 10 l of the relaxin-like factor in
l 50 mM DTT in 3 M guanidinium chloride, 0.2 M
15 Tris, HCI at pH 8.5 for 1 h at 37 C, diluted with 30
l of solvent A, followed by separation on Aquapore
300 (see: Analytical HPLC for conditions).
Upon reduction two chains were generated,
isolated, and the subsequent sequence analyses of both
20 chains showed the desired structure.
UV Spectroscobv: The confirmation and
structure of the synthesized protein was then
confirmed by UV spectroscopy. Such spectroscopy was
performed on an OLIS Cary 15 spectrophotometer
conversion (On-Line Instrument Systems Inc., Bogart,
GA). UV-spectroscopy was used to determine the
protein concentrations of the RLF protein. The
specific absorption coefficient (Er/6=1.40 cm2/mg) was
obtained by direct comparison of UV absorbance and the
recovery of amino acids after hydrolysis and amino
acid analysis.
No partial hydrolysis at the acid labile Asn-Pro
bond was detected.
Circular Dichroism: Further confirmation of
the synthesized protein's identity was performed by CD
spectroscopy, as performed on a Jasco J-710
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spectropolarimeter using a cell of 0.02 cm path
length. Proteins were dissolved in 25 mM Tris/HCI at
pH 7.7 and concentrations were determined by UV
spectroscopy: 0.67 mg/ml for porcine relaxin, 0.54
mg/ml for relaxin-like factor, and 0.55 mg/mi for
human relaxin. Spectra were measured at a resolution
of 0.2 nm, a band width of 2 nm, and 5 spectra were
averaged. Molar ellipticity was calculated according
to Adler et al. (9) using mean residual weights of
110.4 for relaxin-like factor, 113.6 for porcine
relaxin,and 112.5 for human relaxin.
Comparative measurements of circular dichroic
spectra suggested near identity of the solution
structures of RLF and porcine relaxin. See, FIG. 4.
Mass Spectrometry: Finally, mass spectra
were recorded on a JEOL HX110/HX110 4 sector tandem
mass spectrometer (JEOL, Tokyo, Japan) to verify the
protein's identity and proper synthesis. Samples were
dissolved in 0.1% trifluoroacetic acid at a
concentration of about 0.8 nmol/ l.
Mass spectrometry showed the correct mass ion for
the synthetic RLF (found: 6294.6, theoretical 6293.2).
6.2. Production Of Labelled RLF
125I-labeled RLF, containing side chain-
protected tryptophan and methionine, may be prepared
according to the above procedure wherein the
synthesized peptide (10 g in 5 l of water) is then
placed into a 200 l Eppendorf vial and 5 l phosphate
buffer (250 mM, pH 7.4), followed by 2 l of 'uI-(l
mCi), and 5 l of chloramine T (2 mg/ml in phosphate
buffer pH 7.4) are added. The reaction was performed
for 1 min on ice, quenched by addition of 5 l of
sodium thiosulfate (5 H20) (50 mg/mi in phosphate
buffer pH 7.4), and 5 l of NaI (20 mg/ml in phosphate
buffer pH 7.4). The side chain-protecting group of
Trp was removed by addition of 5 l of piperidine.
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After 2 min at room temperature the reaction was
quenched by the addition of 5 l of glacial acid, the
reaction mixture was diluted with 10 l of water and
loaded onto a Aquapore 300 column for separation. The
protein was detected by UV absorbance and peaks were
manually collected into 100 l of 1t bovine serum
albumin in water.
The labelled RLF may be used as an RLF tracer
which could then be used to separate by HPLC the
different RLF derivatives to yield a carrier-free
tracer. See, FIG. 5. Alternatively, such labelled
RLF may also be used in binding assays and for RLF
receptor mapping.
6.3. Receptor Binding Assay
Insulin-receptor binding assays were
performed on crude membrane preparations of term
placenta, as described in Hock and Hollenberg, 1980,
J. Biol. Chem. 255:10731-10736, using luI-iodo-Tyr''ia
porcine insulin as tracer according to the method of
Linde, et al., 1986, J. Chromatogr. 369:327-339.
The assays were performed in HMS-buffer (25 mM
HEPES, 104 mM NaCl, 5 mM MgC121 0.2% bovine serum
albumin; pH 7.4) in a total volume of 100 l. Labeled
insulin (50,000 cpm/assay, 150 pM) and variable
amounts of insulin were incubated with crude membranes
for 1 h at room temperature. Thereafter 1 ml of
buffer was added, the membranes collected by
centrifugation in a microcentrifuge at 14,000 rpm for
5 min, the supernatant discarded, and the tip of the
Eppendorf vial cut off and counted in a7-counter
(Minigamma, LKB,Sweden). To determine nonspecific
binding unlabeled insulin was used at a concentration
of 2 l/ml (0.33 M) and nonspecific binding was
usually below 10% of the total binding.
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Contrary to prior art speculation, RLF does not
bind with any significant degree to the insulin
receptor.
6.4. Relaxin-Binding Assays
Relaxin-binding assays were performed as
described in Yang, et al., 1992, Endocrinology
130:179-185 and BUllesbach, et al., 1994, Endocrine.
Z:1115-1120, using crude membrane preparations of
mouse tissue. Mouse brains of 2 mice were collected
into 15 ml of chilled buffer (25 mM HEPES, 0.14 M
NaCl, 5.7 mM KCI, 0.2 mM phenylmethylsulfonyl-
fluoride, and 80 mg/mi soybean trypsin inhibitor, pH
7.5) supplemented with sucrose (0.25 M, final
concentration). The tissue was homogenized on ice for
10 s with a Polytron homogenizer (Brinlmann, Westbury,
NY) at setting 5. The homogenate was centrifuged at
700 rpm for 10 min at 4 C and the supernatants were
recentrifuged at 10,000 x g for 1 h. The pellet was
resuspended in 15 ml of ice cold binding buffer, 25 mM
HEPES, 0.14 M NaCl, 5.7 mM KCI, 0.2 mM
phenylmethylsulfonylfluoride,and 80 mg/ml soybean
trypsin inhibitor, pH 7.5, supplemented with 1% bovine
serum albumin, and centrifuged for 1 h at 10,000 x g.
The crude membrane preparation was suspended in 1 ml
of binding buffer and 40 l was used per assay. The
assay was performed using 40 l of tracer (about
100,000 cpm of porcine relaxin tracer = 150 pM) and 20
l of relaxin at various concentrations. The assay
was incubated for 1 h at room temperature, and the
suspension diluted with 1 ml of wash buffer (25 mM
HEPES, 0.14 M NaCl, 5.7 mM KCI, 1% bovine serum
albumin, 0.01% NaN3) and centrifuged in Eppendorf
centrifuge at 14,000 rpm for 10 min. The supernatant
was discarded, the tip of the vial cut and counted in
a 7-counter. Nonspecific binding was determined in
the presence of 2 l/ml of unlabeled competitor (0.33
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M). In a typical experiment the specific binding was
between 25% and 40% of the total binding.
Tissue specificity was determined using crude
membrane preparations of leg muscles, kidneys, liver.
brain,and uterus (of estrogen primed mice). The crude
membranes were prepared as described for relaxin.
Binding was based on protein concentration determined
by Lowry.
According to the above-described assay, a
hundred-fold excess of human RLF displaces 50% of the
relaxin tracer from a mouse brain relaxin receptor
preparation. The difference in affinity is still
within the range of specific binding, i.e., several
orders of magnitude better than the binding of insulin
or guinea pig relaxin to this receptor (see generally,
BUlZesbach, et al., 1994, Endocrine. 2:1115-1120)
indicating that RLF recognizes the relaxin receptor.
As discussed above, this result was surprising
because the critical Arg XXX Arg sequence in RLF is
offset toward the C-terminal end of the B chain by
exactly one turn of the helix (See, FIG. 1).
Of the tissues tested with 125I-RLF as tracer such
as brain, uterus, skeletal muscle, kidney and liver,
only the brain- and the uterus membrane preparation
showed specific binding (See, FIG. 6). These are
tissues that also bind relaxin in a competitive and
saturable manner. To test for crossreactivity the
assays were performed with tracers and competitive
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cold molecules exchanged. The results from such
assay are set forth below at Table 1:
TABLE 1
Relaxin-Binding Assay Results
Tissue Tracer Competitor 50% Range
(mouse) Binding
Brain pRLX hRLX 1 ng 0.1-2.0
Brain pRLX hRLF 200 ng 190-220
Uterus pRLX hRLX 1 ng 0.1-2
Uterus pRLX hRLF 10000 n --
Uterus hRLF hRLX 800 ng 600-1000
Uterus hRLF hRLF 0.3 ng 0.1-0.6
Brain hRLF hRLX 1000 ng 900-1100
Brain hRLF hRLF 0.3 ng 0.1-0.6
pRLX = porcine relaxin, hRLX = human relaxin,
pRLF = porcine RLF and hRLF = human RLF
These results suggest strongly that RLF does have its
own receptor in these tissues and that the relaxin
receptor is recognized by RLF, but with a
significantly lower affinity than relaxin.
Furthermore, the data support that the brain and
uterine relaxin receptors differ with respect to
crossreactivity. Specifically, the uterine relaxin
receptor barely recognizes RLF whereas the brain
receptor shows moderate crossreactivity. In general,
the RLF receptor binds its substrate with greater
affinity than the relaxin receptor displays toward
relaxin.
6.5. Sperm Motility Assay
Relaxin and proteins having relaxin-like
activity may be identified by a sperm motility assay.
MateriaZs And Methods. Semen is obtained by
masturbation from healthy volunteers. The sample is
allowed to liquefy at room temperature and is then
mixed with Minimum Essential Medium (MEM) with Hepes
buffer added. This medium is used because it
coincides with that washing medium employed by the in
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vitro clinic at MUSC. The sperm is then separated
from the seminal fluid and MEM by centrifugation. The
resultant sperm pellet is then resuspended in MEM at
room temperature. Aliquots are then place in
siliconized centrifuge tubes and one of several
compounds added: 1)human relaxin 10 ng/ml, 2)human
relaxin 100 ng/ml, 3)relaxin-like factor 10 ng/ml,
4)relaxin-like factor 100 ng/ml, 5)one fraction of
alkaline gland fluid from stingrays diluted 1:8 with
pentoxyfyline. The additive is mixed well with the
sperm/medium mixture. Samples are taken at 0, 2, 4, 6
and 24 hour intervals for automated determination of
the following parameters: 1)motility, 2)progressivity,
3)path velocity, 4)progressive velocity, 5)track
speed, 6)elongation, 7)lateral displacement, 8)cross
beat frequency, 9)straightness, 10)linearity. Briefly
described, each sample is loaded into a Maker heated
specimen chamber and viewed in a light microscope
equipped with laser doppler optics (IVOS, Beverly,
MA). Sample readings taken at approximately 3 minutes
and results are displayed in hard copy form.
Experimental Results. Human relaxin and RLF
added to washed human sperm preserve the motility
compared to untreated controls in which motility and
thus potential fertilizing capacity significantly
declines over time. There was essentially no
difference between the high and low doses of relaxin
in their effects on motility. RLF was as potent in
maintaining sperm motility at both doses as was
relaxin. The most striking effect of both compounds
occurred at 4 hours when motility remained the same or
increased from the previous time period.
Both relaxin and relaxin-like factor were given
in combination to determine if there was an additive
effect. No such effect was observed.
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All the above compounds were compared against
stingray alkaline gland fluid (ALG) from the HPLC.
AGF significantly increased then maintained sperm
motility for the first two time periods then was
approximately 10% higher for the last three time
periods.
6.6. In Vitro Inhibition Of Collagen and
Fibronectin Expression By Human Lung
Fibroblasts
Whether RLF inhibits collagen and
fibronectin expression has been studied in the context
of human lung fibroblasts. Specifically, RLF (1-
l00ng/ml) was applied to human lung fibroblasts in
serum-free medium and assayed for collagen secretion
by biosynthetic labelling with 3H-proline in the
presence of ascorbate and B-aminopropionitrile. When
tested on lung fibroblasts stimulated with TGF-fl,
RLF's ability to inhibit collagen expression at
various dose levels can be determined. The presence
of another extracellular matrix molecule, fibronectin,
in conditioned media was assessed by Western
immunoblotting using an anti-fibronectin polyclonal
antibody, as well as biosynthetic labeling.
6.7. In Vitro Inhibition Of Collagen and
Fibronectin Expression By Synovial
Fibroblasts
Trauma to the shoulder or surgical
intervention of large joints is often associated with
limitation in mobility, in many cases due to an
exaggerated fibrotic response to synovial or capsular
tissue. Extracellular matrix-producing cells, such as
synovial fibroblasts, are capable of the extremes of
degradation or repair. The overproduction of
collagens, fibronectin and other extracellular matrix
molecules can be due to the local expression of
cytokines, such as transforming growth factor TGF-B.
To the extent it has been demonstrated that relaxin
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can decrease TGF-13-stimulated collagen expression in a
dose-dependent manner, up to 30% at a relaxin does of
l0ong/mi and fibronectin expression by 30%, RLF is
also implicated in the modulation and control of
collagen and fibronectin expression.
To test such hypothesis (to determine RLF's
ability to down-regulate collagen and fibronectin
expressed by synovial fibroblasts), fibroblasts can be
explanted from pieces of rheumatoid synovium and
treated with TGF-beta (ing/ml) to stimulate expression
of types I and III collagens. TGF-beta upregulated
collagen expression at the protein level, as measured
by biosynthetic labelling with 3H-proline
incorporation. More specifically, the following
experiments were conducted to determine RLF's ability
to modulate the expression of collagen, fibronectin
and procollegenase in human synovial fibroblasts:
6.7.1. Assay To Determine The Inhibition
ot Collagen Expression
The method for detecting and
measuring collagen formation in the presence of
relaxin described in Unemori and Amento, 1990, J.
Biol. Chem. 265:10681-685 has been modified as follows
to determine the ability of RLF, in vitro, to modulate
the expression of collagen.
Materials And Methods. Rheumatoid synovial
fibroblasts (Strain No. RSF64) were seeded at a
density of 6.25 x 104 cells/cmZ in tissue culture
dishes in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% fetal bovine serum. After 24
hours, the cells were washed and treated with DMEM
supplemented with 0.2% lactalbumin hydroxylsate with
relaxin, RLF and/or transforming growth factor (TGF-
9) =
The cells were simultaneously biosynthetically
labelled with 3H=proline (25 Ci/ml) in the presence of
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ascorbate and BAPN. After 24 hours, the conditioned
media were collected and electrophoresed on 4-12%
polyacrylamide gels (NOVEX) under reducing conditions.
Gels were enhanced, dried, and exposed to X-ray films
for 1-2 weeks. Collagen bands were identified on the
X-ray films as bacterial collagenase-sensitive,
proline-incorporating bands between 95-200 kDa. Band
density was quantified by scanning densitometry and
used as estimates of collagen expression.
Experimental Results. Using the above protocol,
it was determined that RLF decreases collagen
expression independently. Specifically, fibroblast
treatment with TGF-a increased collagen expression by
3.75 fold over that expressed by untreated
fibroblasts. Subsequent addition of RLF (100 ng/ml)
to the TGF-0-treated fibroblasts decreased TGF-0-
stimulated collagen expression by 17%. In comparison,
addition of relaxin (100 ng/ml) decreased TGF-fl-
stimulated collagen expression by 9%.
It was further determined that RLF and relaxin
together synergistically decrease collagen expression.
Specifically, treatment of TGF-0-stimulated cells with
RLF (100 ng/ml) and relaxin (100 ng/ml) decreased
collagen expression by 39%.
6.7.2. Assay To Determine The Inhibition
Of Fibronectin Expression
The method for detecting and
measuring collagen formation in the presence of
relaxin described in Unemori and Amento, 1990, J.
Biol. Chem. 265:10681-685 has been modified as follows
to determine the ability of RLF, in vitro, to modulate
the expression of fibronectin.
MateriaZs And Methods. Specifically, using the
method described in section 6.7.1., the fibronectin
band was identified by size (220 kDa), bacterial
collagenase-resistance, and positive staining using
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commercially available polyclonal anti-fibronectin
antibody (Promega). The fibronectin band was scanned
densitometrically to estimate levels of expression.
Experimental Results. Using the above protocol,
it was determined that RLF decreases fibronectin.
expression independently. Specifically, addition of
RLF (100 ng/ml) to the TGF-fl-treated fibroblasts
decreased TGF-/3-stimulated fibronectin expression by
17%.
6.8. In Vitro Stimulation of Procollagense
Expression By Synovial Fibroblasts
A method for detecting and measuring
procollagenase formation is described in Unemori, et
al., 1991, J. Biol. Chem. 266:23477-482. Such method
was modified to measure the expression of
procollagenase in the presence of RLF as follows:
MateriaZs And Methods. Rheumatoid synovial
fibroblasts (Strain No. RSF112) were seeded at a
density of 6.25 x 104 cells/cm2 in tissue culture
dishes in DMEM supplemented with 10% fetal bovine
serum. After twenty-four hours, the cells were washed
and treated with DMEM supplemented with 0.2%
lactalbumin hydroxylate with relaxin at 1, 10 and 100
ng/ml for 48 to 72 hours. Conditioned media were
collected and an aliquot analyzed by gelatin
zymography. Procollagenase was identified as a
gelatinlytic doublet at 52/57 kDa. The intensity of
the doublet (i.e., the amount of procollagenase
expressed, was quantified by scanning densitometry.
Experimental Results. RLF stimulated expression
of procollagenase in a dose-dependent manner
comparable to that induced by relaxin. RLF at 1, 10
and loo ng/ml stimulated procolleganse expression by
0, 2.0 and 4.2-fold. Relaxin induced procollagenase
expression by 0, 1.6 and 4.9-fold at the equivalent
doses.
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6.9. Cyclic AMP-Release Bioassay
The cAMP assay is a competitive immunoassay
commercially available through Amersham Corporation.
Mater.ials and Methods. To determine cAMP release
induced by RLF, normal human endometrial cells are
grown at 1.2 X 104 cells/well in a 96-well plate in
DMEM/F12 + 10% newborn calf serum. 24 hours later,
the cells are washed in serum-free medium comprised of
DMEM/F12+ 0.2% lactalbumin hydrolysate. 24 hours
later, the cells are treated with relaxin and/or RLF
in the presence of isobutylmethylxanthine and
forskolin for 30 min. The cell lysates are harvested
with 0.1N HCL, neutralized with 0.1N NaOH, then
assayed in the immunoassay (Amersham Corp).
Experimental Results. When relaxin was assayed
at 0.78ng/ml, 86 pM cAMP was measured in endometrial
cell lysates. When RLF (2.5 ug/ml) was simultaneously
added, 470pM cAMP, roughly a 5-fold enhancement in
cAMP production, was measured. When a relaxin
concentration of 3.12ng/ml was tested with and without
RLF (2.5ug/ml), a 2-fold enhancement was measurable
with relaxin + RLF as compared with relaxin alone.
6.10. Mouse Symphysis Pubis Assay
Mouse interpubic ligament assays were
performed essentially as described by Steinetz, et
al., 1960, Endocrinology 67:102-115. Ovariectomized
virgin female mice were printed with 5 g estrogen
cypionate in 100 l sesame oil. Five days later the
mice were injected subcutaneously with human relaxin,
RLF, or mixtures of human relaxin and RLF in 100 l of
0.1% benzopurpurin 4B. Specifically, groups of five
animals received either human relaxin at a suboptimal
dose or a mixture of 0.2 g, 0.4 g and 0.8 g human
relaxin and 5 g of RLF as given in FIG. 8. For
negative control 100 l of 0.1% benzopurpurin 4B in
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water were injected. After 16 hours the mice were
killed in an atmosphere of C02, the symphysis pubis
dissected free, and the distance between the
interpubic bones measured with a dissecting microscope
fitted with transilluminating fiber optics.
The RLF significantly increased the activity of
human relaxin in the mouse bioassay. Increasing RLF
concentrations in the presence of 0.5 pg of human
relaxin showed that 5 pg of RLF was optimal (FIG. 9).
Again the effect of the RLF is clearly recognized. In
the next assay relaxin alone, RLF alone, and a maximal
dose of both are compared (FIG. 7). While RLF alone
had no effect, the relaxin effect at maximal dose was
still augmented by RLF.
The present invention is not to be limited in
scope by the exemplified embodiments which are
intended as illustrations of single aspects of the
invention, and methods which are functiorlally
equivalent are within the scope of the invention.
Indeed, various modifications of the invention in
addition to those described herein will become
apparent to those skilled in the art from the
foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of
the appended claims.
In addition, the publications listed below are of
interest in connection with various aspects of the
invention:
1. Adham, et al., 1993, J. Biol. Chem.
268:26668-26672;
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2. Adler, et al., 1973, Methods Enzymol.
2-Z:675-735;
3. Biillesbach, et al., 1994, Endocrine. 2:1115-
1120;
4. BUllesbach and Schwabe, 1994, J. Biol. Chem.
269:13124-13128;
5. BUllesbach and Schwabe, 1993, Biochem.
Biophys. Res. Commun. 196:311-319;
6. BUllesbach and Schwabe, 1992, J. Biol. Chem.
267:22957-22960;
7. BUllesbach and Shcwabe, 1991, J. Biol. Chem.
266:10754-10761;
8. BUllesbach, et al., 1980, Hoppe Seyler's Z.
Physiol. Chem. 361:865-873;
9. Burkhardt, et al., 1994, Genomics 20:13-19;
10. Eddie et al., 1986, Lancet 1:1344-1346;
11. Eigenbrot, et al., 1991, J. Mol. Biol.
2,1:15-21;
12. Ellman, 1959, Arch. Biochem. Biophys. 82:70-
77;
13. Hock and Hollenberg, 1980, J. Biol. Chem.
255:10731-10736;
14. Linde, et al., 1986, J. Chromatogr. 369:327-
339;
15. Loumaye et al., 1978, Gynecologic and
Obsteric Investigation 9:262-267;
16. Olefsky, et al., 1982, Ann. NY Acad. Sci.
380:200-216;
17. Rembiesa, et al., 1993, Endocrine J. 1:263-
268;
18. Schwabe and BUllesbach, 1994, FASEB J. 8:1-
2;
19. Schwabe and Harmon, 1978, Biochem. Biophys.
Res. Commun. 84:374-380;
20. Sherwood et al., 1980, Endocrinology
107:691-698;
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21. Sherwood and Crnekovic, 1979, Endocrinology
104:893-897;
22. Sieber, et al., 1977, Helv. Chim. Acta
60=27-37;
23. Steinetz, et al., 1960, EndocrinoZogy
67=102-115;
24. Tam, et al., 1991, J. Am. Chem. Soc.
113:6657-6662;
25. Tashima, et al., 1995, J. C1in. Endocrinal.
Metab. 80:707-710; and
26. Yang, et al., 1992, Endocrinology 130:179-
185.
20
30
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